The focus of this proposal will be on the role of protein phosphorylation in neurosecretion. Previous studies have shown that protein III (protein Illa, M/r III) and protein IIIb, M/r 74,000, and protein IIIb, M 55,000, are collectively referred to as protein III) is phosphorylated in response to synaptic activation in intact nerve cell preparations. Moreover, this protein is localized in presynaptic terminals in association with synaptic vesicles. ln a recent series of studies, protein III was shown to be present in adrenal chromaffin cells. In addition, these studies also demonstrated that acetylcholine (ACh) and other secretagogues produce an increase in the phosphorylation of protein III, as well as two other phosphoproteins (87kD, a substrate for protein kinase C; 100 kD, a substrate for calcium/calmodulin- dependent protein kinase III). The increase in protein III phosphorylation was well correlated with the increase in catecholamine release produced by these secretagogues. The goal of the proposed studies is to provide substantive information about the physiological role played by protein phosphorylation, particularly protein III phosphorylation, in catecholamine release from chromaffin cells. The first set of studies will include an analysis of the particular site(s) phosphorylated on protein III as well as a detailed examination of the stoichiometry of the ACh- stimulated phosphorylation the site(s). The second set of studies will be devoted to the determination of the distribution of protein III and other selected phosphoproteins (87kD, 100 kD) in subcellular fractions prepared from the adrenal medulla and from purified chromaffin cells. The last set of studies will be devoted to developing and implementing the technology for microinjecting macromolecules into primary cultures of chromaffin cells. The ultimate goal of these studies is to obtain direct evidence about the physiological role of protein phosphorylation in neurosecretion using the chromaffin cell as a model system.